Effect of
wzx
(
rfbX
) Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis in
Pseudomonas aeruginosa
O5
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ABSTRACT
The
wbp
cluster of
Pseudomonas aeruginosa
O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly
wbpF
), contiguous with
wzy
in the
wbp
cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or “flippase.” Insertion of a Gm
r
cassette at two separate sites within the putative
wzx
gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal
wzx
gene replacement mutations. Surprisingly, inactivation of
wzx
also led to a marked delay in production of the ATP-binding cassette–transporter-dependent,
d
-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in
trans
. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in
P. aeruginosa
O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103–1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of
wzx
mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.
